EP0755513A1

EP0755513A1 - Method and apparatus for electrophoretic analysis

Info

Publication number: EP0755513A1
Application number: EP95916890A
Authority: EP
Inventors: Bengt-Göran Andersson
Original assignee: Pharmacia Biotech AB
Current assignee: Cytiva Sweden AB
Priority date: 1994-04-14
Filing date: 1995-04-12
Publication date: 1997-01-29
Also published as: WO1995028636A1; CA2187780A1; SE9401251D0; JPH09512102A

Abstract

In a method and system for electrophoretic analysis of fluorophore-labelled susbtance mixtures, an electrophoresis zone (1) having at least one lane (3), and photometric detector means (4) fixed relative to this lane for detecting separated fluorophore-labelled substances as they pass the detector means (4) are used. According to the invention, there are further used means (5, 8, 9, 10) for alternately emitting to the electrophoresis lane light of different wavelengths or wavelength bands (μ1, μ2) corresponding to the excitation wavelengths for two or more different fluorophores, and synchronisation means for relating a detected fluorescent light emission to a respective excitation wavelength.

Description

METHOD AND APPARATUS FOR ELECTROPHORETIC ANALYSIS

The present invention relates to electrophoretic analysis, and more particularly to a method and an apparatus for electrophoretic separation of fluorescence- labelled mixtures of substances, particularly nucleic acid fragments obtained in sequencing reactions .

Since some time past there are two basic methods for DNA sequence determination, viz. the chemical degradation method (Maxam and Gilbert, Proc. nat. Acad. Sci. U.S.A., Vol. 74, p. 560-564 (1977)) and the chain termination method (Sanger et al., Proc. nat. Acad. Sci. U.S.A., Vol. 74, p. 5463-5467 (1977)) .

In the chemical degradation method, the DNA strand to be analysed is labelled at one end with a detectable tag. The sample is divided into four parts, each of these parts being treated with a respective reagent capable of cleaving specifically at one of the four bases. The reaction conditions are adapted to obtain approximately one or a few cleavages per molecule. Each reaction mixture will then contain a mixture of a number of fragments of different lengths, among them end-labelled fragments of lengths corresponding to all possible cleavage sites, i.e. the base that is specifically cleaved by the respective reagent. By separating the four reaction mixtures according to fragment size in parallel in four lanes on an electrophoretic gel, a detectable ladder of labelled bands representing the relative positions of one of the four bases is obtained. From these fragment ladders the DNA sequence in question may then directly be read.

In the chain termination method, which is the one most used today, the DNA fragment to be analysed is instead used as a template for DNA synthesis in four different reaction mixtures by means of a starter sequence, or so-called primer, hybridised to the 5 ' -end of the strand, and DNA polymerase in the presence of the four deoxynucleoside triphosphates. Each reaction mixture contains a low concentration of a chain terminator in the form of a respective one of the four dideoxynucleoside triphoshate analogues which upon incorporation in the growing chain prevents continued growth. Either the primer, one or more of the deoxynucleoside triphosphates or the terminators are labelled with a detectable tag. In each reaction mixture is then obtained a population of partially synthesized labelled DNA molecules having a common 5 ' -end but varying in length to a base-specific 3' -end. Electrophoretic separation of the four different reaction mixtures side by side on a gel as in the Maxam-Gilbert method gives four fragment ladders from which the desired DNA sequence thus may be read.

While originally, radioactive phosphorus was used as label and the fragment ladders obtained after the electrophoresis were imaged on autoradiograms, the use of fluorescent labels has made possible more or less automatic analysis with continuous detection of the fragment bands that migrate in the different lanes using fluorescence detectors. US-A-4, 675, 095 describes an advantageous electrophoretic apparatus for this purpose where the exciting light is introduced sideways between the two glass plates that hold the electrophoretic gel between them, and the emitted fluorescence is detected on one side of the gel perpendicularly to the excitation light path. By such an arrangement a single light source may be used for a number of electrophoresis lanes, simultaneously as background light caused by fluorescence and light scattering in the glass plates themselves are avoided. A commercial automated development of this type of apparatus for DNA sequence analysis based on the chain termination method and using labelling with one and the same fluorescent tag, or fluorophore, is marketed by Pharmacia Biotech AB, Uppsala, Sweden, under the trade name A.L.F. DNA Sequencer™ (where A.L.F. stands for "Automated Laser Fluorescent") . That apparatus has 40 electrophoresis lanes where the excitation is effected by laser light and the light emitted from the fluorophore-containing DNA fragment bands is detected by an equal number of separate fixed detectors (photodiodes) , one for each lane. After the samples have been loaded onto the gel, the detection signals are collected automatically and sent to a computer for storing and processing. Since each sample requires four lanes, i.e. one lane for each of the four different base- related terminators, 10 different DNA samples may thus be analysed at the same time.

The object of the present invention is to increase the capacity of DNA sequencing apparatus of this type by making it possible to analyse two or more samples simultaneously in one and the same electrophoretic gel lane. According to the invention this is achieved by labelling these samples with different fluorophores excited at different wavelengths by means of separate excitation sources . By having the excitation/detection of the respective fluorophores take place at different times, the different sample-related signals may be distinguished from one another. The inventive concept is, however, not restricted to DNA sequencing but may equally well be applied to any electrophoretic separation, not only of nucleic acids but also of other types of substances, such as e.g. proteins. One aspect of the invention therefore relates to a method of electrophoretically analysing a mixture of fluorophore-labelled substances by detecting the substances in an electrophoresis lane as they are separated and pass photometric detector means fixed relative to the electrophoresis lane, which method is characterized by simultaneously analysing in the same electrophoresis lane two or optionally more different substance mixtures which are labelled by different fluorophores capable of being excited at different wavelengths or wavelength bands by alternately illuminating the detection area with light of these different wavelengths, and relating a detected fluorescent light emission to the respective fluorophore on the basis of the time for the excitation thereof. Another aspect of the invention relates to a system for electrophoretic analysis of fluorophore-labelled substance mixtures, comprising an electrophoresis zone having at least one lane, and photometric detector means fixed relative to this lane for detecting separated fluorophore-labelled substances as they pass the detector means, which system is characterized by further comprising means for alternately emitting to said lane light of different wavelengths or wavelength bands corresponding to the excitation wavelengths of two or more different fluorophores, and synchronisation means for relating a detected fluorescent light emission to a respective excitation wavelength.

In a preferred embodiment the method and system of the invention are adapted to nucleic acid sequencing. While the "samples" labelled with different fluorophores in the present case are primarily meant to consist of fragment populations obtained by one and the same base-specific sequencing reaction of the above-mentioned type on several different DNA or RNA fragments to be analysed, the differently labelled samples in question may, of course, also be two or more different fragment populations obtained by base-specific sequencing reactions on one and the same DNA or RNA fragment to be analysed. Also so-called fragment analysis may advantageously be performed with the method and system according to the invention.

In the following the invention will be described in more detail with regard to a specific, non-limiting embodiment, reference being made to the accompanying drawings, wherein

Fig. 1 is a schematic illustration of a prior art system for nucleic acid sequencing,

Fig. 2 is a schematic illustration of an embodiment of a system according to the invention, and Fig. 3 is a schematic diagram which shows the filter characteristic of a filter for filtering out fluorescent light from the detector in the system of Fig. 2. The prior art analytical system shown in Fig. 1 is of the type mentioned above with A.L.F. DNA Sequencer™ (Pharmacia Biotech AB, Uppsala, Sweden) as a commercial example and intended for a fluorescent label. It operates with a fixed laser beam and a number of fixed detectors arranged across the separation gel. To this end the illustrated system has an electrophoresis unit 1 having a gel 2 with a number of lanes, here as example four lanes, each represented by a detection zone 3 monitored by a respective photodetector 4 (only one shown) in the form of a photodiode. A laser 5 introduces a light beam 6 of wavelength λ-]_ into the gel plane 2 for excitation of the fluorescent label of the DNA fragments that pass the detection zones 3. Emitted fluorescence is detected perpendicularly to the gel plane by the photodiodes 4. An optical filter 7 prevents the excitation light from reaching the detectors 4 while simultaneously allowing emitted fluorescence to pass through. The detectors 4 are meant to be connected to a computer-based data collecting system so that the DNA sequence determined in each case may be presented in chart form.

In Fig. 2, the system of Fig. 1 has been modified according to the invention for the use of two different fluorophores, the capacity of the system thereby being doubled. In comparison with the system in Fig. 1 this modified system has in addition to the laser 5 an extra laser 8 which emits light at another wavelength λ2 adapted to excite the additional fluorophore.

Further, a rotating chopper disc 9, or "chopper", is disposed in the light path of the beams from the two lasers 5, 8, so that the beams are chopped up by alternately being allowed to pass through the chopper disc. A dichroic mirror 10 arranged to reflect the beam from the laser 5 of wavelength λ_]_ and transmit the beam from the laser 8 of wavelength λ2 is placed between the chopper disc 9 and the electrophoretic apparatus 1. Hereby the two laser beams are combined and aligned to the same physical path, i.e. the same optical axis, for a common incidence into the gel 2. For the above-mentioned automatic data collecting system to know when the respective lasers 5 and 8 are active, the detector electronics is synchronized with the laser chopping through sync pulses from the chopper disc 9. The chopping or pulsing of the laser beams may, of course, be accomplished in other ways. For example, the rotating disc and the dichroic mirrors may be combined to a single component that provides for both chopping and beam combination by replacing the rotating disc by a mechanical component of equivalent construction where mirrors have been substituted for the solid parts of the chopper disc. Further, in order to avoid moving mechanics, acousto- optical or electro-optical modulators may be used instead of the rotating disc. It is, of course, also possible to accomplish an analogous function with solid state components.

In the modified system according to Fig. 2, the optical filter 7 in Fig. 1 is replaced by a filter 11 capable of blocking both laser wavelengths λ]_ and λ2 instead of only the wavelength λ^. One embodiment of such a filter consists of a combination of a glass type absorption filter and an interference filter (thin film filter) , the absorption filter functioning as a substrate for the interference filter. An example of a filter characteristic obtained hereby (transmission versus wavelength) is shown in Fig. 3. As a specific example, such a filter with damping at laser wavelengths 488 n (argon laser) and 633 ran (He-Ne-laser or laser diode) has been obtained from Omega Optical Inc., U.S.A., which on behalf of applicant deposited an RB (rejection band) type interference filter on an OG 515 filter glass with high damping for 488 nm to also obtain a high damping for a band around 633 nm. Exemplary of a suitable fluorophore combination for these laser wavelengths is fluorescein, which exhibits a light absorption maximum at a wavelength of about 490 nm and a light emission maximum at a wavelength of about 530 nm, and Cy5 (Biological Detection Systems, Inc., Pittsburgh, PA, U.S.A.), which is a carbocyanine-based dye containing a reactive succinimidyl ester and which exhibits a light absorption maximum at a wavelength of about 650 nm and a light emission maximum at about 670 nm.

With the analytical system shown in Fig. 2, a doubled capacity is thus obtained in comparison with the prior art system according to Fig. 1. The capacity may, of course, be further increased if three, or optionally even more lasers/fluorophores are used.

The invention is, of course, not restricted to the embodiments specifically described above and shown in the drawings, but many variations and modifications may be made within the scope of the general inventive concept as defined in the following claims.

Claims

1. A method of electrophoretically analysing a mixture of fluorophore-labelled substances by detecting the substances in an electrophoresis lane as they are separated and pass photometric detector means fixed relative to the electrophoresis lane, characterized by simultaneously analysing in the same electrophoresis lane two or optionally more different substance mixtures which are labelled by different fluorophores capable of being excited at different wavelengths or wavelength bands by alternately illuminating the detection area with light of said different wavelengths, and relating a detected fluorescent light emission to the respective fluorophore on the basis of the time for the excitation thereof.

2. The method according to claim 1, characterized in that said substance mixtures comprise fluorophore-labelled nucleic acid fragments .

3. The method according to claim 2 , characterized in that said fluorophore-labelled nucleic acid fragments are DNA fragments obtained in sequencing reactions, particularly according to the chain termination method.

4. The method according to claim 3 , characterized in that at least two different reaction mixtures from different analytical samples, which reaction mixtures are specific for the same base, are run in each electrophoresis lane.

5. The method according to claim 3, characterized in that at least two different reaction mixtures from the same analytical sample, which reaction mixtures are specific for the same base, are run in each electrophoresis lane.

6. A system for electrophoretic analysis of fluorophore- labelled substance mixtures, comprising an electrophoresis zone (1) having at least one lane (3) , and photometric detector means (4) fixed relative to said lane for detecting separated fluorophore-labelled substances as they pass the detector means (4) , characterized in that the system further comprises means (5, 8, 9, 10) for alternately emitting to said lane light of different wavelengths or wavelength bands (λi , λ2) corresponding to the excitation wavelengths of two or more different fluorophores, and synchronisation means for relating a detected fluorescent light emission to a respective excitation wavelength.

7. The system according to claim 6, characterized in that said light of different wavelengths is directed into the electrophoresis zone along one and the same optical axis.

8. The system according to claim 7, characterized in that the system comprises at least two lasers (5, 8) and a chopping and beam combining device (9, 10) arranged between the lasers and the electrophoresis zone.

9. The system according to claim 8, characterized in that the chopping and beam combining device comprises a rotating chopper disc (9) and a dichroic mirror (10) .

10. The system according to any one of claims 6 to 9, characterized in that the system comprises filter means (11) designed to selectively prevent excitation light of at least two different wavelengths from reaching the detector means (4) .

11. The system according to claim 10, characterized in that said filter means (11) comprises a combination of absorption filter and interference filter.